PRI-724 and IWP-O1 Wnt Signaling Pathway Inhibitors Modulate the Expression of Glycolytic Enzymes in Tongue Cancer Cell Lines.

The dysregulation of energetic metabolism is one of the hallmarks of cancer cells. Indeed, the growth of head and neck squamous cell carcinoma (HNSCC) cells depends heavily on glycolytic activity, which can be considered a potential therapeutic target. Wnt signaling is one of the pathways that undergoes upregulation in HNSCC. Our previous studies have shown that Wnt signaling inhibitors-PRI-724 and IWP-O1-attenuate tongue SCC survival and reduce glucose uptake and lactate release. The aim of this research was to further evaluate the possible mechanisms of the previously observed effects. We assessed the effect of PRI-724 and IWP-O1 on the expression of selected glycolytic enzymes: phosphofructokinase M, pyruvate kinase M2, and lactate dehydrogenase. Relative transcript expression was assessed by real-time PCR, and protein levels by Western blot. Moreover, clinical data concerning mRNA and protein expression, gene promoter methylation, and HNSCC patients' survival time were analyzed by the UALCAN tool, and protein-protein interaction was assessed using the STRING database. Experimental and bioinformatic data confirmed the relation between Wnt signaling and glycolytic enzymes in tongue cancer cells and HNSCC clinical samples. Overall, the inhibition of glucose metabolism by Wnt signaling inhibitors is a promising mode of action against tongue cancer cells.

Combinations of PRI-724 Wnt/ß-Catenin Pathway Inhibitor with Vismodegib, Erlotinib, or HS-173 Synergistically Inhibit Head and Neck Squamous Cancer Cells.

The Wnt/ß-catenin, EGFR, and PI3K pathways frequently undergo upregulation in head and neck squamous carcinoma (HNSCC) cells. Moreover, the Wnt/ß-catenin pathway together with Hedgehog (Hh) signaling regulate the activity of cancer stem cells (CSCs). The aim of this study was to investigate the effects of the combinatorial use of the Wnt/ß-catenin and Hh pathway inhibitors on viability, cell cycle progression, apoptosis induction, cell migration, and expression of CSC markers in tongue (CAL 27) and hypopharynx (FaDu) cancer cells. Co-inhibition of Wnt signaling with EGFR or PI3K pathways was additionally tested. The cells were treated with selective inhibitors of signaling pathways: Wnt/ß-catenin (PRI-724), Hh (vismodegib), EGFR (erlotinib), and PI3K (HS-173). Cell viability was evaluated by the resazurin assay. Cell cycle progression and apoptosis induction were tested by flow cytometric analysis after staining with propidium iodide and Annexin V, respectively. Cell migration was detected by the scratch assay and CSC marker expression by the R-T PCR method. Mixtures of PRI-724 and vismodegib affected cell cycle distribution, greatly reduced cell migration, and downregulated the transcript level of CSC markers, especially POU5F1 encoding OCT4. Combinations of PRI-724 with erlotinib or HS-173 were more potent in inducing apoptosis.

The combinatorial inhibition of Wnt signaling and Akt kinase is beneficial for reducing the survival and glycolytic activity of tongue cancer cells.

BACKGROUND: Wnt signaling is important in the development of head and neck squamous cell carcinomas (HNSCC); however, Wnt pathway inhibitors lack satisfactory potency when used in monotherapy. The aim of this study was to assess the effects of the combinations of Wnt-signaling inhibitors and the inhibitor of Akt kinase on the survival and glycolytic activity of tongue carcinoma cells. METHODS: CAL27, SCC-25, and BICR22 tongue cancer cell lines were used. Cells were treated with Wnt signaling (PRI-724 and IWP-O1) and Akt-kinase inhibitors. The effect of the chemicals on cell viability and cytotoxicity were evaluated by MTS and CellTox Green assays, respectively. Cell cycle distribution was analyzed cytometrically after propidium iodide staining. Annexin V binding to externalized phosphatidylserine and analysis of mitochondrial potential allowed the assessment of apoptosis. Glucose uptake and lactate release were evaluated luminometrically. Additionally, the viability of cells in spheroids was analyzed based on ATP content. RESULTS: The Akt-kinase inhibitor showed significant cytotoxicity toward primary cancer cells. Moreover, its pro-apoptotic effects were enhanced by Wnt-pathway inhibitors. The activity of Akt inhibitor was even higher (by twofold) in 3D spheroids in comparison to cells grown in monolayer. The synergistic reduction in the growth of spheroids was observed between Akt inhibitor and IWP-O1. Reduced glucose consumption may play a part in the combinatorial effects of these chemicals. CONCLUSION: The results point to the therapeutic potential of the combinatorial use of Wnt inhibitors together with Akt inhibitors in HNSCC.

The Wnt Signaling Pathway Inhibitors Improve the Therapeutic Activity of Glycolysis Modulators against Tongue Cancer Cells.

Excessive glucose metabolism and disruptions in Wnt signaling are important molecular changes present in oral cancer cells. The aim of this study was to evaluate the effects of the combinatorial use of glycolysis and Wnt signaling inhibitors on viability, cytotoxicity, apoptosis induction, cell cycle distribution and the glycolytic activity of tongue carcinoma cells. CAL 27, SCC-25 and BICR 22 tongue cancer cell lines were used. Cells were treated with inhibitors of glycolysis (2-deoxyglucose and lonidamine) and of Wnt signaling (PRI-724 and IWP-O1). The effects of the compounds on cell viability and cytotoxicity were evaluated with MTS and CellTox Green tests, respectively. Apoptosis was evaluated by MitoPotential Dye staining and cell cycle distribution by staining with propidium iodide, followed by flow cytometric cell analysis. Glucose and lactate concentrations in a culture medium were evaluated luminometrically. Combinations of 2-deoxyglucose and lonidamine with Wnt pathway inhibitors were similarly effective in the impairment of oral cancer cells' survival. However, the inhibition of the canonical Wnt pathway by PRI-724 was more beneficial, based on the glycolytic activity of the cells. The results point to the therapeutic potential of the combination of low concentrations of glycolytic modulators with Wnt pathway inhibitors in oral cancer cells.

(+)-Usnic acid modulates the Nrf2-ARE pathway in FaDu hypopharyngeal carcinoma cells.

Naturally occurring phytochemicals of different origin and structure, arctigenin, bergenin, usnic acid and xanthohumol, were shown to affect Nrf2 pathway in the context of various diseases, but their effect on this pathway in cancer cells was not extensively investigated. This study aimed to evaluate the effect of these compounds on Nrf2 expression and activation in hypopharyngeal FaDu squamous cell carcinoma cells. FaDu cells were treated with 2 or 10 µM arctigenin, bergenin, (+)-usnic acid or xanthohumol for 24 h. While arctigenin, bergenin, and xanthohumol did not affect either Nrf2 expression or activation, (+)-usnic acid treatment increased its transcript level and increased the nuclear/cytosol Nrf2 protein ratio-the measure of Nrf2 pathway activation. Consequently, (+)-usnic acid enhanced the transcription and translation of Nrf2 target genes: NQO1, SOD, and to a lesser extent, GSTP. The treatment of FaDu cells with (+)-usnic acid decreased both GSK-3ß transcript and protein level, indicating its possible involvement in Nrf2 activation. All the tested compounds decreased Bax mRNA but did not change the level of Bax protein. (+)-Usnic acid tended to increase the percentage of early apoptotic cells and LC3 protein, autophagy marker. Significant induction of p53 also was observed after treatment with (+)-usnic acid. In summary, the results of this study indicate that low concentrations of (+)-usnic acid activate Nrf2 transcription factor, most probably as a result of ROS accumulation, but do not lead to FaDu hypopharyngeal carcinoma cells death.

Lichen-Derived Depsides and Depsidones Modulate the Nrf2, NF-κB and STAT3 Signaling Pathways in Colorectal Cancer Cells.

The study aimed to evaluate the possible modulation of Nrf2, NF-ĸB and STAT3 signaling pathways in the colorectal cancer (CRC) cells line DLD-1 and HCT116 by secondary metabolites of lichens. An attempt was made to indicate the most promising targets in these signaling pathways. Attention was also paid to the effects of the compounds tested on CRC cells using anakoinosis-that is, simultaneous analysis of several signaling pathways. The effects of the tested natural compounds on the activity of selected transcriptional factors related to CRC were analyzed by Western blot and RT-PCR assays. The highest activity against CRC cells was shown by physodic and salazinic acids from the studied secondary metabolites of lichens. As a result, an increase in the activation of transcription factor Nrf2 and the expression of its selected target genes was observed. Physodic and salazinic acids induced the opposite effect in relation to the NF-κB and STAT3 pathways. These results confirmed our earlier observations that lichen-derived compounds have the ability to modulate signaling pathway networks. While caperatic acid affected Wnt/ß-catenin to the most extent, salazinic acid was the most potent modulator of Nrf2, NF-κB and STAT3 pathways. Physodic acid seemed to affect all the investigated pathways.

Oleanolic acid oxime derivatives and their conjugates with aspirin modulate the NF-κB-mediated transcription in HepG2 hepatoma cells.

The aim of this study was to evaluate the effect of new oleanolic acid oxime (OAO) derivatives and their conjugates with aspirin (ASP) on the expression and activation of NF-κB in human hepatoma HepG2 cells. OAO derivatives showed a stronger cytotoxic effect against HepG2 cells compared with their conjugates with aspirin. Moreover, conjugation of OAO with ASP led to enhanced downregulation of NF-κB expression and activation. Among the hybrids with ASP, compounds: 19, 3-(2-acetoxy)benzoyloxyiminoolean-12-en-28-oic acid morpholide and 13, 3-(2-acetoxy)benzoyloxyiminoolean-12-en-28-oic acid methyl ester, differing, respectively, in morpholide and methyl ester groups at the C-17 position of oleanolic acid (OA) molecule were the most efficient. COX-2 transcript and protein levels were also diminished after treatment with these compounds. The results of this study indicate that the new derivatives of OAO and particularly their conjugates with ASP, downregulate the expression of COX-2 in HepG2 cells by modulating the NF-κB signaling pathway and suggest their potential application in the prevention of liver inflammation and cancer.

Lichen-derived caperatic acid and physodic acid inhibit Wnt signaling in colorectal cancer cells.

Lichens are a source of secondary metabolites which possess important biological activities, including antioxidant, antibacterial, anti-inflammatory, and cytotoxic effects. The anticancer activity of lichens was shown in many types of tumors, including colorectal cancers (CRC). Several studies revealed that the application of lichen extracts diminished the proliferation of CRC cells and induced apoptosis. Colon carcinogenesis is associated with aberrations in Wnt signaling. Elevated transcriptional activity of ß-catenin induces cell survival, proliferation, and migration. Thus, the inhibition of Wnt signaling is a promising therapeutic strategy in colorectal cancer. The aim of this study was the evaluation of the effects of lichen-derived depsides (atranorin, lecanoric acid, squamatic acid) and depsidones (physodic acid, salazinic acid) and a poly-carboxylic fatty acid-caperatic acid, on Wnt signaling in HCT116 and DLD-1 colorectal cancer cell lines. HCT116 cells were more sensitive to the modulatory effects of the compounds. PKF118-310, which was used as a reference ß-catenin inhibitor, dose-dependently reduced the expression of the classical ß-catenin target gene-Axin2 in both cell lines. Lecanoric acid slightly reduced Axin2 expression in HCT116 cells while caperatic acid tended to reduce Axin2 expression in both cell lines. Physodic acid much more potently decreased Axin2 expression in HCT116 cells than in DLD-1 cells. Physodic acid and caperatic acid also diminished the expression of survivin and MMP7 in a cell line and time-dependent manner. None of the compounds affected the nuclear translocation of ß-catenin. This is the first report showing the ability of caperatic acid and physodic acid to modulate ß-catenin-dependent transcription.

Prognostic significance of the methylation of Wnt pathway antagonists-CXXC4, DACT2, and the inhibitors of sonic hedgehog signaling-ZIC1, ZIC4, and HHIP in head and neck squamous cell carcinomas.

OBJECTIVES: Aberrations in Wnt and Shh signaling pathways are related to the pathogenesis of head and neck carcinomas, and their activation frequently results from epigenetic alterations. This study aimed to assess the frequency of methylation of negative regulators of Wnt signaling: CXXC4, DACT2, HDPR1, and FBXW11 and Shh signaling: HHIP, PTCH1, SUFU, ZIC1, and ZIC4 and correlate it with clinicopathological features in this group of patients. MATERIALS AND METHODS: Methylation-specific PCR was used to detect gene promoter methylation, and real-time PCR was used to assess gene expression level. RESULTS: The analysis of the occurrence of gene promoter methylation in head and neck carcinoma cell lines indicated that CXXC4, DACT2, HHIP, ZIC1, and ZIC4 are methylated in these tumors. These genes were further analyzed in tumor sections from oral and laryngeal cancer patients. Gene methylation rate was higher in laryngeal tumors. The methylation index in tumor samples correlated with the overall survival in a subgroup of oral cancer patients who died of the disease. Moreover, ZIC4 methylation correlated with lymph node involvement in oral cancer patients. CONCLUSIONS: Our findings corroborate that the activation of Wnt signaling in head and neck squamous cell carcinoma (HNSCC) is related to epigenetic silencing of its negative regulators. Moreover, the results indicate that the same mechanism of activation may operate in the case of Shh signaling. CLINICAL RELEVANCE: The methylation of ZIC4 may be considered a new prognostic marker in oral cavity and oropharyngeal tumors. Further investigations should determine the diagnostic significance of methylation of ZIC4, HHIP, and DACT2 in head and neck carcinomas.

The methylation of a panel of genes differentiates low-grade from high-grade gliomas.

Epigenetic changes play an important role in the pathogenesis of gliomas and have the potential to become clinically useful biomarkers. The aim of this study was the evaluation of the profile of promoter methylation of 13 genes selected based on their anticipated diagnostic and/or prognostic value. Methylation-specific PCR (MSP) was used to assess the methylation status of MGMT, ERCC1, hMLH1, ATM, CDKN2B (p15INK4B), p14ARF, CDKN2A (p16INK4A), RASSF1A, RUNX3, GATA6, NDRG2, PTEN, and RARß in a subset of 95 gliomas of different grades. Additionally, the methylation status of MGMT and NDRG2 was analyzed using pyrosequencing (PSQ). The results revealed that the methylation index of individual glioma patients correlates with World Health Organization (WHO) tumor grade and patient's age. RASSF1A, RUNX3, GATA6, and MGMT were most frequently methylated, whereas the INK4B-ARF-INK4A locus, PTEN, RARß, and ATM were methylated to a lesser extent. ERCC1, hMLH1, and NDRG2 were unmethylated. RUNX3 methylation correlated with WHO tumor grade and patient's age. PSQ confirmed significantly higher methylation levels of MGMT and NDRG2 as compared with normal, non-cancerous brain tissue. To conclude, DNA methylation of a whole panel of selected genes can serve as a tool for glioma aggressiveness prediction.

The negative regulators of Wnt pathway-DACH1, DKK1, and WIF1 are methylated in oral and oropharyngeal cancer and WIF1 methylation predicts shorter survival.

The deregulation of Wnt signaling has recently emerged as one of the drivers of head and neck cancers. This is frequently related to the methylation of several antagonists of this pathway. This study aimed at the assessment of the profile of methylation of Wnt pathway antagonists and the determination of the prognostic value of the methylation of selected genes in oral carcinomas. The methylation of DACH1, DKK1, LKB1, PPP2R2B, RUNX3, SFRP2, and WIF-1 was analyzed in 16 oral squamous cell carcinoma cell lines using the methylation-specific polymerase chain reaction. The methylation of selected genes was further analyzed in tumor sections from 43 primary oral carcinoma patients. The analysis of oral carcinoma cell lines showed very frequent methylation of SFRP2 and WIF-1 and also a less frequent methylation of DACH1 and DKK1. On the other hand, RUNX3 was methylated only in one cell line, while LKB1 and PPP2R2B were not methylated in any of the cell lines. The biallelic methylation of DKK1 correlated with the low level of expression of this gene. Further evaluation of the methylation of DACH1, DKK1, and WIF1 in a clinical patient group confirmed the frequent methylation of WIF1 and intermediate or low frequency of methylation of DACH1 or DKK1, respectively. Importantly, the methylation of WIF-1 correlated with shorter survival in oral cancer patients. Overall, the methylation of the antagonists of Wnt pathway is frequently detected in oral squamous cell carcinomas. The methylation of WIF1 may be considered a prognostic marker in oral cancers.

Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin.

Oral and oropharyngeal cancers are characterized by relatively low 5- year survival rates due to many factors, including local recurrence. The identification of new molecular markers may serve for the estimation of prognosis and thus augment treatment decisions and affect therapy outcome. The aim of this study was to describe the morphological characteristics and the DNA methylation status of the CDKN2A,CDH1, ATM, FHIT and RAR- genes in the central and peripheral part of the tumor and the surgical margin and evaluate their prognostic significance. 53 patients with oral and oropharyngeal cancer were enrolled to the prospective study, and had been primarily treated surgically. Correlations between morphological data, hypermethylation status and clinicopathological data, as well as prognosis, were assessed. Nuclei polymorphism highly correlated with T stage (p < 0.0001), N stage (p < 0.046), and metastases to the lymph nodes pN (p < 0.004 ). Also, the number of cells in irregular mitosis correlated with T stage (p < 0.004), and highly with pN (p < 0.009). The significance of CDKN2A hypermethylation as a good prognostic factor was also established in the Kaplan-Meir test. The ultrastructural analysis showed that none of the examined tumors had homogenous texture and that resection margin specimens clean in HE stained tissue samples frequently contained single tumor cells or few cells in groups surrounded by connective tissue. This indicates the superiority of electron microscopy over standard histopathological analysis. Thus, a combination of such morphological examination with epigenetic parameters described herein could result in the discovery of promising new prognostic markers of the disease.

Frequent hypermethylation of WNT pathway genes in laryngeal squamous cell carcinomas.

BACKGROUND: Aberrations in the function of the WNT signaling pathway have been recently implicated in the pathogenesis of head and neck cancer, and the hypermethylation of several WNT cascade inhibitors were shown to be useful in disease prognosis. However, the extent of deregulation of WNT pathway by DNA hypermethylation has not been studied in detail in laryngeal cancer so far. The aim of this study was to establish the frequency of methylation of WNT pathway negative regulators in laryngeal squamous cell carcinomas and evaluate its prognostic significance. METHODS: Twenty-six laryngeal squamous cell carcinoma cell lines and samples obtained from twenty-eight primary laryngeal carcinoma patients were analyzed. The methylation status of DKK1, LKB1, PPP2R2B, RUNX3, SFRP1, SFRP2, and WIF-1 was assessed using the methylation-specific polymerase chain reaction. RESULTS: Frequent hypermethylation of DKK1, PPP2R2B, SFRP1, SFRP2, and WIF-1 was detected, and a high methylation index was usually observed. Half of the cell lines analyzed and seventy percent of primary laryngeal carcinoma cases were characterized by the methylation of at least four genes. The hypermethylation of PPP2R2B or WIF-1 was associated with longer survival in laryngeal carcinoma cell lines. Moreover, the concurrent methylation of PPP2R2B and SFRP1 differentiated primary from recurrent laryngeal carcinoma cell lines. CONCLUSIONS: Frequent hypermethylation of WNT pathway negative regulators is observed in laryngeal squamous cell carcinomas. The possible prognostic significance of the methylation of DKK1, PPP2R2B, and SFRP1 needs to be evaluated in further prospective studies.

Hawthorn (Crataegus oxyacantha L.) bark extract regulates antioxidant response element (ARE)-mediated enzyme expression via Nrf2 pathway activation in normal hepatocyte cell line.

Hawthorn (Crataegus oxyacantha L.), a plant used in traditional medicine, is a rich source of procyanidins which have been reported to exhibit antioxidant and anti-carcinogenic activity. In this study, we assessed the effect of hawthorn bark extract (HBE) on Nrf2 pathway activation in THLE-2 and HepG2 cells. Treatment with 1.1 µg/mL, 5.5 µg/mL and 11 µg/mL of HBE resulted in the translocation of Nrf2 from the cytosol to the nucleus in both cell lines; however, the accumulation of phosphorylated Nrf2 was observed only in THLE-2. Accordingly, treatment of cells with HBE was associated with an increase in the mRNA and protein level of such Nrf2-dependent genes as glutathione S-transferases (GSTA, GSTP, GSTM, GSTT), NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) (0.2-1.1-fold change, p < 0.05), however, only in normal THLE-2 hepatocytes. The induction of NQO1 correlated with an increased level of p53 (0.21-0.42-fold change, p < 0.05). These effects may be related to induction of phosphorylation of upstream ERK and JNK kinases. Collectively, the results suggest that the Nrf2/ARE pathway may play an important role in the regulation of procyanidin-mediated antioxidant/detoxifying effects in hepatocytes, and this may explain the hepatoprotective and chemopreventive properties of these phytochemicals.

Triple Combinations of Histone Lysine Demethylase Inhibitors with PARP1 Inhibitor-Olaparib and Cisplatin Lead to Enhanced Cytotoxic Effects in Head and Neck Cancer Cells.

PARP inhibitors are used to treat cancers with a deficient homologous recombination (HR) DNA repair pathway. Interestingly, recent studies revealed that HR repair could be pharmacologically impaired by the inhibition of histone lysine demethylases (KDM). Thus, we investigated whether KDM inhibitors could sensitize head and neck cancer cells, which are usually HR proficient, to PARP inhibition or cisplatin. Therefore, we explored the effects of double combinations of KDM4-6 inhibitors (ML324, CPI-455, GSK-J4, and JIB-04) with olaparib or cisplatin, or their triple combinations with both drugs, on the level of DNA damage and apoptosis. FaDu and SCC-040 cells were treated with individual compounds and their combinations, and cell viability, apoptosis, DNA damage, and gene expression were assessed using the resazurin assay, Annexin V staining, H2A.X activation, and qPCR, respectively. Combinations of KDM inhibitors with cisplatin enhanced cytotoxic effects, unlike combinations with olaparib. Triple combinations of KDM inhibitors with cisplatin and olaparib exhibited the best cytotoxic activity, which was associated with DNA damage accumulation and altered expression of genes associated with apoptosis induction and cell cycle arrest. In conclusion, triple combinations of KDM inhibitors (especially GSK-J4 and JIB-04) with cisplatin and olaparib represent a promising strategy for head and neck cancer treatment.

Anti-Aging Properties of Chitosan-Based Hydrogels Rich in Bilberry Fruit Extract.

Photoaging is a process related to an increased level of reactive oxygen species (ROS). Polyphenols can scavenge free radicals in the body, which can delay skin aging. Therefore, our work aimed to prepare a biologically active extract from dry fruits of Vaccinium myrtillus or Vaccinium corymbosum and use it for the preparation of hydrogels for topical application. Therefore, eight different extracts (using V. myrtillus and V. corymbosum and different extraction mixtures: methanol, methanol-water 1:1, water, acetone-water 1:1) were prepared and their phytochemical (total polyphenolic content, total flavonoid content, total anthocyanin content) and biological properties (antioxidant, anti-hyaluronidase, and anti-tyrosinase activity) were assessed. Cytotoxicity towards HaCaT keratinocytes was also determined. Based on the results, the acetone-water extract from V. myrtillus was selected for further study. Using the Design of Experiments approach, chitosan-based hydrogels with bilberry fruit extract were prepared. The content of extract and chitosan were selected as independent factors. The activity of hydrogels depended on the extract content; however, the enzyme-inhibiting (anti-hyaluronidase and anti-tyrosinase) activity resulted from the presence of both the extract and chitosan. Increased concentration of chitosan in the hydrogel base led to increased viscosity of the hydrogel and, consequently, a slower release of active compounds. To get optimal hydrogel characteristics, 1% extract and 2.5% MMW chitosan were utilized. The research suggests the validity of using bilberry fruit extracts in topical preparations with anti-aging properties.

Towards effectiveness of cell free DNA based liquid biopsy in head and neck squamous cell carcinoma.

Liquid biopsy is a minimally invasive procedure, that uses body fluids sampling to detect and characterize cancer fingerprints. It is of great potential in oncology, however there are challenges associated with the proper handling of liquid biopsy samples that need to be addressed to implement such analysis in patients' care. Therefore, in this study we performed optimization of pre-analytical conditions and detailed characterization of cfDNA fraction (concentration, length, integrity score) in surgically treated HNSCC patients (n = 152) and healthy volunteers (n = 56). We observed significantly higher cfDNA concentration in patients compared to healthy controls (p < 0.0001) and a time dependent decrease of cfDNA concentration after tumor resection. Our results also revealed a significant increase of cfDNA concentration with age in both, healthy volunteers (p = 0.04) and HNSCC patients (p = 0.000002). Moreover, considering the multitude of HNSCC locations, we showed the lack of difference in cfDNA concentration depending on the anatomical location. Furthermore, we demonstrated a trend toward higher cfDNA length (range 35-10380 and 500-10380 bp) in the group of patients with recurrence during follow-up. In conclusion, our study provide a broad characterization of cfDNA fractions in HNSCC patients and healthy controls. These findings point to several aspects necessary to consider when implementing liquid biopsy in clinical practice including: (I) time required for epithelial regeneration to avoid falsely elevated levels of cfDNA not resulting from active cancer, (II) age-related accumulation of nucleic acids accompanied by less efficient elimination of cfDNA and (III) higher cfDNA length in patients with recurrence during follow-up, reflecting predominance of tumor necrosis.

Betanin, a beetroot component, induces nuclear factor erythroid-2-related factor 2-mediated expression of detoxifying/antioxidant enzymes in human liver cell lines.

Our recent study has shown that beetroot juice protects against N-nitrosodimethylamine (NDEA)-induced liver injury and increases the activity of phase II enzymes, suggesting the activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway. The aim of the present study was to further explore the mechanism of the activity of beetroot by evaluating the cytoprotective effects of its major component. The influence of betanin (BET) on the activation of Nrf2 and the expression of GSTA, GSTP, GSTM, GSTT, NQO1 and HO-1 was assessed in two hepatic cell lines: non-tumour THLE-2 and hepatoma-derived HepG2 cell lines. The level of the tumour suppressor p53 in both cell lines and the methylation of GSTP in HepG2 cells were also evaluated. Treatment of both cell lines with 2, 10 and 20 µm of BET resulted in the translocation of Nrf2 from the cytosol to the nucleus. The mRNA and nuclear protein levels of Nrf2 and the binding of Nrf2 to ARE sequences were increased only in the THLE-2 cells and were accompanied by the phosphorylation of serine/threonine kinase (AKT), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). BET also significantly increased the mRNA and protein levels of GSTP, GSTT, GSTM and NQO1 in these cells. Conversely, besides the translocation of Nrf2 from the cytosol to the nucleus, BET did not modulate any of the other parameters measured in the HepG2 cells. BET did not change the methylation of GSTP1 in these cells either. These results indicate that BET through the activation of Nrf2 and subsequent induction of the expression of genes controlled by this factor may exert its hepatoprotective and anticarcinogenic effects. Moreover, the activation of mitogen-activated protein kinases may be responsible for the activation of Nrf2 in the THLE-2 cells.

Targeting cancer stem cells as a strategy for reducing chemotherapy resistance in head and neck cancers.

PURPOSE: Resistance to chemotherapy and radiotherapy is the primary cause of a poor prognosis in oncological patients. Researchers identified many possible mechanisms involved in gaining a therapy-resistant phenotype by cancer cells, including alterations in intracellular drug accumulation, detoxification, and enhanced DNA damage repair. All these features are characteristic of stem cells, making them the major culprit of chemoresistance. This paper reviews the most recent evidence regarding the association between the stemness phenotype and chemoresistance in head and neck cancers. It also investigates the impact of pharmacologically targeting cancer stem cell populations in this subset of malignancies. METHODS: This narrative review was prepared based on the search of the PubMed database for relevant papers. RESULTS: Head and neck cancer cells belonging to the stem cell population are distinguished by the high expression of certain surface proteins (e.g., CD10, CD44, CD133), pluripotency-related transcription factors (SOX2, OCT4, NANOG), and increased activity of aldehyde dehydrogenase (ALDH). Chemotherapy itself increases the percentage of stem-like cells. Importantly, the intratumor heterogeneity of stem cell subpopulations reflects cell plasticity which has great importance for chemoresistance induction. CONCLUSIONS: Evidence points to the advantage of combining classical chemotherapeutics with stemness modulators thanks to the joint targeting of the bulk of proliferating tumor cells and chemoresistant cancer stem cells, which could cause recurrence.

Natural products modulating epigenetic mechanisms by affecting histone methylation/demethylation: Targeting cancer cells.

Many natural products can exert anticancer or chemopreventive activity by interfering with the cellular epigenetic machinery. Many studies indicate the relevance of affecting DNA methylation and histone acetylation, however the influence on the mechanisms related to histone methylation are often overlooked. This may be associated with the lagging evidence that changes in the action of histone methylation writers and erasers, and subsequent alterations in the profile of histone methylation are causally related with carcinogenesis. Recent animal studies have shown that targeting histone methylation/demethylation affects the course of experimentally induced carcinogenesis. Existing data suggest that numerous natural compounds from different chemical groups, including green tea polyphenols and other flavonoids, curcuminoids, stilbene derivatives, phenolic acids, isothiocyanates, alkaloids and terpenes, can affect the expression and activity of crucial enzymes involved in the methylation and demethylation of histone lysine and arginine residues. These activities have been associated with the modulation of cancer-related gene expression and functional changes, including reduced cell proliferation and migration, and enhanced apoptosis in various cancer models. Most studies focused on the modulation of the expression and/or activity of two proteins - EZH2 (a H3K27 methyltransferase) and LSD1 (lysine demethylase 1A - a H3K4/9 demethylase), or the effects on the global levels of histone methylation caused by the phytochemicals, but data regarding other histone methyltransferases or demethylases are scarce. While the field remains relatively unexplored, this review aims to explore the impact of natural products on the enzymes related to histone methylation/demethylation, showing their relevance to carcinogenesis and cancer progression.